A. Lysostaphin
Lysostaphin is a potent antimicrobial agent first identified in Staphylococcus simulans (formerly known as S. staphylolyticus). Lysostaphin is a bacterial endopeptidase capable of cleaving the specific cross-linking polyglycine bridges in the cell walls of staphylococci, and is therefore highly lethal thereto. Expressed in a single polypeptide chain, lysostaphin has a molecular weight of approximately 27 kDa.
The cell wall bridges of Staphylococcus aureus, a coagulase positive staphylococcus, contain a high proportion of glycine, therefore lysostaphin is particularly effective in lysing S. aureus. Lysostaphin has also demonstrated the ability to lyse Staphylococcus epidermidis. 
S. aureus is a highly virulent human pathogen. It is the cause of a variety of human diseases, ranging from localized skin infections to life-threatening bacteremia and infections of vital organs. If not rapidly controlled, a S. aureus infection can spread quickly from the initial site of infection to other organs. Although the foci of infection may not be obvious, organs particularly susceptible to infection include the heart valves, kidneys, lungs, bones, meninges and the skin in burn patients.
Staphylococcal infections, such as those caused by S. aureus, are a significant cause of morbidity and mortality, particularly in settings such as hospitals, schools, and infirmaries. Patients particularly at risk include infants, the elderly, the immunocompromised, the immunosuppressed, and those with chronic conditions requiring frequent hospital stays.
Patients at greatest risk of acquiring staphylococcal infections, are those undergoing inpatient or outpatient surgery, in the Intensive Case Unit (ICU), on continuous hemodialysis, with HIV infection, with AIDS, burn victims, people with diminished natural immunity from treatments or disease, chronically ill or debilitated patients, geriatric populations, infants with immature immune systems, and people with intravascular devices.
U.S. Pat. No. 6,028,051 to Climo, et al., discloses a method for the treatment of staphylococcal disease. Relatively high doses of lysostaphin of at least 50 and preferably 100 milligrams of lysostaphin per kilogram of body weight are used for treatment. The relatively high doses of lysostaphin can be used in single dose treatments or multiple dose treatments. The lysostaphin analog can be used singularly or in combination with additional antibiotic agents. The '051 patent also discloses that the cloning and sequencing of the lysostaphin gene permits the isolation of variant forms of lysostaphin that can have properties similar to or different from those of wild type lysostaphin.
U.S. Pat. No. 6,315,996 to O'Callaghan, discloses a method for using lysostaphin as an effective antibiotic for topical treatment of staphylococcus corneal infections. U.S. Pat. No. 5,760,026 to Blackburn et al., discloses a method for using lysostaphin to eliminate and cure staphylococcal infections including the cure of mastitis by intramammary infusion. The method is directed to use in dairy cows.
However, small proteins (less than about 70 kDa), such as lysostaphin, have a relatively short half-life in blood after intravenous injection. Lysostaphin's rapid clearance from circulation may reduce its efficacy. At the same time, because it is derived from a bacterial species and therefore foreign to any mammalian species, lysostaphin is also a very immunogenic molecule, which further stimulates its clearance from the blood stream, especially in subjects that have had previous exposure to lysostaphin. Thus, lysostaphin's short circulating half-life cannot be effectively countered by increasing the amount or frequency of dosage. There exists a need for a means by which the circulating half-life of lysostaphin may be increased without increasing the amount or frequency of administration. It would even be more desirable to increase the circulating half-life of lysostaphin while at the same time reducing the amount or frequency of administration.
B. Polymer Conjugation
The conjunction of biologically active polypeptides with water-soluble polymers such as PEG is well-known. PEGylation is a process in which therapeutic polypeptides, such as enzymes and hormones, are coupled to one or more chains of polyethylene glycol to provide improved clinical properties in terms of pharmacokinetics, pharmacodynamics, and immunogenicity.
PEGylation can alter the characteristics of the polypeptide without affecting the ability of the parent molecule to function, thereby producing a physiologically active, reduced or non-immunogenic, water-soluble polypeptide composition. The polymer protects the polypeptide from loss of activity by reducing its clearance and susceptibility to enzymatic degradation, and the composition can be injected into the mammalian circulatory system with substantially no immunogenic response. PEGylation of enzymes and other polypeptides is described in detail in U.S. Pat. No. 4,179,337 to Davis et al., and in Zalipsky, “Functionalized Poly(ethylene glycol) for Preparation of Biologically Relevant Conjugates,” Bioconjugate Chem., 6, 150-165 (1995), both of which are incorporated by reference in their entirety herein.
Davis et al. disclose that polypeptides modified with PEG have dramatically reduced immunogenicity and antigenicity. PEG conjugates exhibit a wide range of solubilities and low toxicity, and have been shown to remain in the bloodstream considerably longer than the corresponding native compounds, yet are readily excreted. The conjugates have been shown not to interfere with the activity of other enzymes in the bloodstream or the conformation of polypeptides conjugated thereto.
PEG conjugation is typically accomplished by means of two commonly used types of linkages. One type of conjugation reacts a polypeptide amino group with a PEG molecule having an active carbonate, ester, aldehyde or tresylate group. Another type of conjugation reacts a polypeptide thiol group with a PEG molecule having an active vinyl sulfone, maleimide, haloacyl or thiorthopyridyl group, or other suitable electrophile. See, for example, Hermanson, Bioconjugate Techniques (Academic Press, San Diego 1966). One of the two terminal hydroxyls of the PEG is blocked by conversion to an alkoxy group when intermolecular cross-linking is not desired. A PEG molecule with one terminal methoxy group is referred to as mPEG.
The PEG molecule may be linear or branched, whereby PEG conjugates can be created by conjugating a single large PEG moiety to a single conjugation site, a single branched (but smaller) PEG moiety to a single conjugation site, or several small PEG moieties to multiple conjugation sites. When multiple conjugation sites are employed, this can result in the loss of bioactivity. In addition to PEG homopolymers, the polymer molecule can be copolymerized with other alkylene oxide moieties, or it can be another poly(alkylene oxide) homopolymer or copolymer.
A number of PEG-conjugates of therapeutic proteins have been developed exhibiting reduced immunogenicity and antigenicity and longer clearance times, while retaining a substantial portion of the protein's physiological activity. U.S. Pat. No. 4,261,973 describes the PEG conjugation of immunogenic allergen molecules to reduce the immunogenicity of the allergen. U.S. Pat. No. 4,301,144 discloses that the conjugation of PEG to hemoglobin increases the oxygen-carrying ability of the molecule. U.S. Pat. No. 4,732,863 discloses the conjugation of PEG to antibodies to reduce binding to Fc receptors. EP 154,316 and Katre et al., Proc. Natl. Acad. Sci., 84, 1487 (1987) disclose PEG conjugated lymphokines such as IL-2. U.S. Pat. No. 4,847,325 discloses the selective conjugation of PEG to Colony Stimulating Factor-1 (CSF-1).
Interferon-β2 (INF-β2) has been conjugated without a loss of biological activity to the succinimidyl ester of a single, branched PEG molecule consisting of two 20 kDa mono-methoxy PEG chains connected through a lysine molecule via urethane bonds. This PEG conjugate is targeted for the treatment of hepatitis C, by affecting host immunity and enhancing immune clearance of the virus. The administration of the INF-β2 can be reduced to once weekly from three-to-seven times a week, simplifying and improving patient compliance. In addition, serum levels are maintained with minimal peak-to-trough variation, toxicity is reduced, and efficacy is increased.
FDA approved PEGylated therapeutic polypeptides in clinical use include PEG conjugates of INF-β2, adenosine deaminase and asparaginase. PEGylated therapeutic polypeptides awaiting FDA approval include PEG conjugates of IL-2, IL-6 and Tumor Necrosis Factor. Each of these PEGylated products contains a polypeptide targeted at host cell activities or cancerous host cells, but not to microbes.EG conjugation has been disclosed of proteins such as alpha-1-proteinase inhibitor, uricase, superoxide dismutase, streptokinase, plasminogen activator, IgG, albumin, INFβ2, lipoprotein lipase, horseradish peroxidase, catalase and arginase. These proteins also do not target microbes. The PEG conjugation was reported to improve circulating half-life, decrease immunogenicity, increase solubility and, in general, increase efficacy, thereby permitting less frequent dosing. In most cases, the proteins required multiple PEG conjugations per molecule to improve in vivo performance, and the activity in vitro was significantly decreased by such modification.
WO 01/04287 published Jan. 18, 2002, discloses the use of mutagenic processes to modify polypeptides in general, and staphylokinase in particular, for improved performance of the PEG conjugate thereof.
There is otherwise no disclosure of a PEG conjugation of antimicrobial agents to optimize pharmacokinetics and pharmacodynamics. No reference discloses the conjugation of an antimicrobial agent, or a mutagenic modification thereof, with PEG so as to retain its biological activity while also increasing its circulating half-life and efficacy, and decreasing its antibody binding and toxicity.